The methane monooxygenase hydroxylase (MMOH) contains a carboxylate-bridged dinuclear iron center that serves as the active site for the biological oxidation of methane to methanol. Previous X-ray crystallographic studies have resulted in the high resolution structure determination of this protein in both the oxidized diiron(III) and reduced diiron(II) states (Rosenzweig et al., 1993; Rosenzweig et al., 1995). The structure of a second crystal form of the protein has also been determined recently (Rosenzweig et al., 1997). One facet of current structural work is aimed at probing the mechanism by which substrates access the active site, which is buried in the interior of the protein. To approach this objective, crystals were cocrystallized with a number of small substrate and product molecules, and the structures were examined. Questions regarding the mechanism of alkane hydroxylation are also being addressed by studying the interaction of the hydroxylase with suicide substrates. Current theories regarding the mechanism of hydroxylase activity invoke important hydrogen bonding interactions in the active site cavity. MMOH crystals which are typically grown at pH 7.0 were soaked in buffers of either higher or lower pH. Diffraction intensities dropped off rapidly as a function of soaking time at pH 6.0, but data were collected from crystals at pH 8.5. The collective knowledge gained from X-ray diffraction studies of MMOH serves to complement the ongoing mechanistic work in our laboratory.